包装规格：100ml: 一抗50ml+二抗50ml；500ml：一抗250ml+二抗250ml
Product Introduction The Pico ECL substrate can provide a bright signal for western blotting experiments using horseradish peroxidase (HRP) conjugates. The ECL substrate is compatible with various membranes, blocking solutions and a wide range of antibody diluents for outstanding performance , Versatility and high cost performance, to meet the needs of users for western blotting applications. Features of PicoECL substrate: • PicoECL — an enhanced chemiluminescent substrate for horseradish peroxidase (HRP) • Low picogram sensitivity—detect low picogram protein bands on nitrocellulose membrane or PVDF membrane • Long signal duration—Under optimized conditions, the blot strips incubated with the substrate can continuously output a detectable light signal for 6 to 8 hours • Stable reagents — the working solution remains stable within 24 hours; the kit can be stored stably for up to 1 year at room temperature • Economical price-optimized for diluted antibody concentration conditions: • 0.2 to 1.0 µg/mL primary antibody (diluted 1:1,000 to 1:5000 times with 1 µg/mL stock solution) • 10 to 50 ng/mL secondary antibody (diluted 1:20,000 to 1:100,000 times with 1 µg/mL stock solution)
Important hint
1 For best results, all components of the system must be optimized, including sample volume, primary and secondary antibody concentrations, and types of membranes and blocking reagents. 2 The use of this product requires a lower antibody concentration than the use of precipitated colorimetric HRP substrate detection. To optimize the antibody concentration, perform a systematic dot blot analysis. 3 No one blocking reagent is the best for all systems, so it is very necessary to find the most suitable blocking buffer for each western blot detection system. The blocking reagent may cross-react with the antibody, resulting in non-specific signals. The blocking buffer also affects the sensitivity of the system. When switching from one substrate to another, signal attenuation or background increase sometimes occurs. The reason may be that the blocking buffer is not suitable for the new detection system. 4 When using avidin/biotin detection system, avoid using milk as a blocking reagent, because milk contains unquantified endogenous biotin, which will cause high background signals. 5 Ensure the usage volume of washing buffer, blocking buffer, antibody solution, and substrate working solution to ensure that the blotting membrane is completely covered by the liquid during the entire experiment to prevent the membrane from drying out. Increasing the usage of blocking buffer and washing buffer can reduce non-specific signals 6 For best results, use a shaker during the incubation step. 7 Add Tween20 (final concentration 0.05-0.1%) to blocking buffer and diluted antibody solution to reduce non-specific signals. Use high-quality products such as detergents. It is kept in ampoules, and the content of peroxides and other impurities is very low. 8 Do not use sodium azide as a preservative in the buffer. Sodium azide is an inhibitor of HRP 9 Avoid direct contact between your hands and the membrane, wear gloves or use clean tweezers during the experiment 10 All equipment must be clean and free from foreign substances. Metal instruments (such as scissors) must not have visible rust. Rust may cause spot formation and high background. 11 The substrate working solution can be stable for 8 hours at room temperature. Sunlight or any other strong light may damage the substrate. For best results, store the substrate working solution in an amber bottle and avoid long-term exposure to any strong light and short-term exposure to routine laboratory lighting Will not damage the working fluid
Operation overview Note: Optimize the concentration of antigen and antibody. The recommended antibody dilution must be used to ensure a positive result. Please refer to other required materials for the recommended dilution range. 1) Dilute the concentration of the primary antibody to 0.2～1ug/ml 2) Dilute the concentration of the secondary antibody to 10～50ng/mL 3) Mix the two substrate components at a ratio of 1:1 to prepare the substrate working solution. Note: Exposure to sunlight or any other strong light may damage the working fluid. For best results, store this working fluid in an amber bottle and avoid long-term exposure to any strong light. Exposure to routine lighting in the laboratory for a short period of time will not damage the working fluid. 4) Incubate the blotting membrane in West Pico ECL Substrate Working Solution for 5 minutes. 5) Aspirate the excess reagent. Cover the blotting membrane with a clean plastic film. 6) Expose the blot film on the X-ray film.
Other required materials l The blotting membrane that has been transferred: Use a suitable electrophoresis method to separate the proteins, and transfer these proteins to the nitrocellulose membrane. l Dilution buffer: use Tris or phosphate buffer. l Washing buffer: Add 5 mL of 10% Tween-20 to 1000 mL of dilution buffer (the final concentration of Tween-20 will be 0.05%). l Blocking reagent: add 0.5mL of 10% Tween-20 to 100mL of blocking buffer, and select a blocking buffer that has the same basic components as the dilution buffer. l Primary antibody: Select an antibody specific to the target protein. Use dilution buffer to prepare a 1ug/ml stock solution of the antibody. Use blocking reagent to dilute the antibody from the stock solution to the antibody working solution. The dilution is between 1:1000 and 1:5000 or the concentration of the antibody working solution is 0.2～1ug/ml. The best dilution depends on the amount of the primary antibody and the antigen on the membrane. l HRP-labeled secondary antibody: Choose a HRP-labeled secondary antibody that specifically binds to the secondary antibody, and use the dilution buffer to prepare a 1ug/ml stock solution of the antibody. Use blocking reagent to dilute the antibody from the stock solution to the antibody working solution. The dilution is between 1:20000 and 1:100,000 or the concentration of antibody working solution is 10-50ng/ml. This concentration range also applies when using streptavidin-HRP. The optimal dilution of the secondary antibody depends on the HRP-labeled secondary antibody and the amount of antigen on the membrane. l Film cassettes, developing and fixing reagents for processing radiographic films l Rotary shaker used for incubation. Detailed steps of western blotting 1) Take the imprinted membrane out of the protein transfer equipment, add a suitable blocking solution and incubate in the greenhouse for 20-60 minutes while shaking. To block non-specific protein binding sites on the membrane. Please note: it is very important to use the antibody dilution recommended in the previous section. 2) Take the membrane out of the blocking solution and incubate it with the working solution of the primary antibody in the greenhouse for 1 hour while shaking; or incubate overnight at 28°C without shaking. 3) Add sufficient washing buffer to the membrane to ensure that the buffer completely covers the membrane. Incubate with shaking for ≥5 minutes, change the washing buffer and repeat this step 4-6 times. Increasing the volume of the wash buffer, the number of washes and the washing time help to reduce the background signal. Note: Before incubation, a short rinsing of the membrane in the washing buffer will improve the washing efficiency. Please note: It is very important to use the HRP-labeled secondary antibody dilution suggested above. 4) Incubate the HRP-labeled secondary antibody working solution with the membrane in the greenhouse for 1 hour while shaking. 5) Repeat step 3 to remove unbound HRP-labeled secondary antibody. Note: The membrane must be washed thoroughly after incubating with the HRP-labeled secondary antibody. 6) Mix A solution and B solution in equal proportions to prepare a working solution. Use 0.01～0.1ml working solution per cm2 of membrane. The working fluid can be stable for 8 hours in the greenhouse. Note: Heavy rain, sunlight or any other strong light may damage the working fluid. In order to obtain the result of the mouth, keep this working fluid in an amber bottle and avoid any strong light from long-term heavy rain. Common lighting in the laboratory will not harm the working fluid. 7) Incubate the imprint membrane in the working solution for 5 minutes. 8) Take out the imprint film from the working fluid and place it in a plastic sheet or clean plastic paper (film). Use a piece of absorbent paper to absorb the excess liquid, and carefully press out air bubbles between the imprint and the plastic paper . 9) Place the imprint film wrapped in plastic paper (film) in a film cassette with the protein side facing up, and turn off all lights except for the lights suitable for film exposure (such as red safety lights). Note: The film must be kept dry during the exposure. In order to obtain the best results, the following measures should be taken: * Ensure that the excess substrate is completely removed from the film and plastic paper. * Use gloves during the entire film processing period. * Do not place the imprint film on the developed film, because the chemicals on the film will weaken the signal. 10) Place the X-ray film on top of the film. It is recommended that the first exposure is 60 seconds. The exposure time can be adjusted later to achieve the best results. The chemiluminescence reaction is most intense during the first 5-30 minutes after the substrate incubation. This reaction can last for several hours, but the intensity will decrease over time. If the substrate is exposed for a longer time after incubation, the exposure time may need to be extended to obtain a stronger signal. If you use a phosphorescent storage imaging device (such as Bio-Rad's molecular imager system) or a CCD camera, a longer exposure time may be required. Warning: Any movement between film and film may cause artificial non-specific signals on the film. 11) Use appropriate developer and fixer to develop the film. If the signal is too strong, shorten the exposure time or peel off the imprinted film and reduce the antibody concentration to retest. | 
